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3D droplet-based bioprinting technology is an innovative and time-saving additive manufacturing method, which enables spatial patterning of biological materials and biochemical and living cells for multiple clinical and research applications. Understanding the criteria that control droplet spreading behavior during droplet impact is of great importance in controlling printing resolution and optimizing the printing performance. In this experimental work, the spreading of 3D printed cell-laden droplets was studied with side and bottom view images. The droplets contain 1 × 10 7 cells/ml input cell concentration and corresponding Φ = 0.52 % cell volume fraction and impact onto a flat hydrophilic substrate, a pre-printed droplet, and a pre-printed thin liquid film. The cell-laden droplet impact morphology, the maximum spreading factor, and the cell distribution under different printing conditions (89 < W e < 365 , 174 < R e < 414) in a 3D bioprinting process were characterized. It was found that on the hydrophilic flat substrate, the cells homogeneously distributed into a disk structure. The maximum spreading factor, β max , can be well described by the correlation formulas based on the energy balance and volume conservation. A power-law scaling formula was found to describe the maximum spreading in terms of the Weber number for cell-laden droplet impact on both pre-printed droplets and thin liquid films, where β max ∝ W e 0.25 . Input cell concentration, up to 1 × 10 7 cells/ml, was found to have negligible effect on the maximum droplet spreading factor in a 3D bioprinting process. [ABSTRACT FROM AUTHOR]

3D cell bioprinting is an innovative and time-saving additive manufacturing technology; it precisely generates complex cell-laden constructs to overcome the limitations of 2D cell culture and conventional tissue engineering scaffold technology. Many efforts have been made to evaluate the bioprinter performance by considering printed cell number and the consistency of printed cell number. In this paper, a modified droplet imaging system is used to study the printing performance for a micro-valve-based 3D bioprinter using fluorescence MCF-7 cells. The effects of droplet dispensing physics (dosing energy E d), ink properties (Z number—the inverse of the Ohnesorge number and particle sedimentation velocity), and input cell concentration are considered. The droplet imaging system demonstrates a strong capability and accuracy in analyzing bioprinting performance for printed cell density less than 300 cells/droplet. The average printed cell number is positively correlated with the increasing input cell concentration, dosing energy, and printing time. Printing ink, with Z number ranging from 4 to 7.41 and cell sedimentation velocity at 9.45 × 10 − 8 m/s, can provide the estimated printed cell number and consistent cell printing results within 2 min printing time. Printing inks with higher Z number or cell sedimentation velocity should be ejected under dosing energy below 2.1 La and printed right after filling the reservoir to achieve reliable and stable printing results. [ABSTRACT FROM AUTHOR]

Human liver organoids (HLOs) hold significant potential for recapitulating the architecture and function of liver tissues in vivo. However, conventional culture methods of HLOs, forming Matrigel domes in 6-/24-well plates, have technical limitations such as high cost and low throughput in organoid-based assays for predictive assessment of compounds in clinical and pharmacological lab settings. To address these issues, we have developed a unique microarray 3D bioprinting protocol of progenitor cells in biomimetic hydrogels on a pillar plate with sidewalls and slits, coupled with a clear bottom, 384-deep well plate for scale-up production of HLOs. Microarray 3D bioprinting, a droplet-based printing technology, was used to generate a large number of small organoids on the pillar plate for predictive hepatotoxicity assays. Foregut cells, differentiated from human iPSCs, were mixed with Matrigel and then printed on the pillar plate rapidly and uniformly, resulting in coefficient of variation (CV) values in the range of 15–18%, without any detrimental effect on cell viability. Despite utilizing 10–50-fold smaller cell culture volume compared to their counterparts in Matrigel domes in 6-/24-well plates, HLOs differentiated on the pillar plate exhibited similar morphology and superior function, potentially due to rapid diffusion of nutrients and oxygen at the small scale. Day 25 HLOs were robust and functional on the pillar plate in terms of their viability, albumin secretion, CYP3A4 activity, and drug toxicity testing, all with low CV values. From three independent trials of in situ assessment, the IC50 values calculated for sorafenib and tamoxifen were 6.2 ± 1.6 μM and 25.4 ± 8.3 μM, respectively. Therefore, our unique 3D bioprinting and miniature organoid culture on the pillar plate could be used for scale-up, reproducible generation of HLOs with minimal manual intervention for high-throughput assessment of compound hepatotoxicity. [ABSTRACT FROM AUTHOR]

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Multicellular spheroids and organoids are promising in vitro 3D models in personalized medicine and drug screening. They replicate the structural and functional characteristics of human organs in vivo. Microfluidic technology and micro‐nano fabrication can fulfill the high requirement of the engineering approach in the growing research interest in spheroids and organoids. In this review, spheroids and organoids are comparatively introduced. Then it is illustrated how spheroids‐ and organoids‐on‐a‐chip technology facilitates their establishment, expansion, and application through spatial‐temporal control, mechanical cues modeling, high‐throughput analysis, co‐culture, multi‐tissue interactions, biosensing, and bioimaging integration. The potential opportunities and challenges in developing spheroids‐ and organoids‐on‐a‐chip technology are finally outlooked. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) cell agglomerates, such as microtissues, organoids, and spheroids, become increasingly relevant in biomedicine. They can provide in vitro models that recapitulate functions of the original tissue in the body and have applications in cancer research. For example, they are widely used in organ-on-chip systems. Microsensors can provide essential real-time information on cell metabolism as well as the reliability and quality of culture conditions. The combination of sensors and 3D cell cultures, especially single spheroids, is challenging in terms of reproducible formation, manipulation, and access to spheroids, precise positioning near sensors, and high cell-to-volume ratios to obtain meaningful biosignals in the most parallel approach possible. To overcome this challenge, we combined state-of-the-art bioprinting techniques to automatically print tumour spheroids directly into microwells of a chip-based electrochemical oxygen sensor array. We demonstrated highly accurate and reproducible spheroid formation (diameter of approx. 200 μm) and a spheroid deposition precision of 25 μm within a volume of 22 nl per droplet. Microstructures and hydrogel-coated microwells allowed the placement of single MCF-7 breast cancer spheroids close to the sensor electrodes. The microelectrode wells were sealed for oxygen measurements within a 55 nl volume for fast concentration changes. Accurate and stable amperometric oxygen sensor performance was demonstrated from atmospheric to anoxic regions. Cellular respiration rates from single tumour spheroids in the range of 450–850 fmol min−1 were determined, and alterations of cell metabolism upon drug exposure were shown. Our results uniquely combine bioprinting with 3D cell culture monitoring and demonstrate the much-needed effort for facilitation, parallelization, sensor integration, and drug delivery in 3D cell culture and organ-on-chip experiments. The workflow has a high degree of automation and potential for scalability. In order to achieve greater flexibility in the automation of spheroid formation and trapping, we employed a method based on drop-on-demand liquid handling systems, instead of the typical on-chip approach commonly used in microfluidics. Its relevance ranges from fundamental metabolic research over standardization of cell culture experiments and toxicological studies, to personalized medicine, e.g. patient-specific chemotherapy. [ABSTRACT FROM AUTHOR]